Methods of coronary imaging

ABSTRACT

The present invention provides methods of myocardial perfusion imaging and increasing coronary blood flow of a mammal that are accomplished by administering doses of a compound that is a selective partial A 2A  receptor agonist with a short duration of action, in particular regadenoson, useful for, among other indications, myocardial imaging and coronary vasodilation, and determining areas of insufficient blood flow.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/070,768 filed on Mar. 2, 2005, now U.S. Pat. No. 7,582,617, which isa continuation of U.S. patent application Ser. No. 10/614,702, filedJul. 3, 2003, now abandoned, which is a continuation of U.S. patentapplication Ser. No. 09/792,617, filed on Feb. 23, 2001, now abandoned,which claims the benefit of the filing date of Provisional PatentApplication Ser. Nos. 60/184,296, filed Feb. 23, 2000, and 60/219,876,filed Jul. 21, 2000, the specifications of each of which areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to a method of identifying compounds that areselective partial A_(2A)-adenosine receptor agonists, preferably with ashort duration of action. Such compounds provide coronary dilation inmammals without causing corresponding significant peripheralvasodilation. The invention also relates to a method of using suchcompounds as adjuncts in cardiac imaging.

BACKGROUND

Myocardial perfusion imaging (MPI) is a diagnostic technique useful forthe detection and characterization of coronary artery disease. Perfusionimaging uses materials such as radionuclucides to identify areas ofinsufficient blood flow. In MPI, blood flow is measured at rest, and theresult compared with the blood flow measured during exercise on atreadmill (cardiac stress testing), such exertion being necessary tostimulate blood flow. Unfortunately, many patients are unable toexercise at levels necessary to provide sufficient blood flow, due tomedical conditions such as peripheral vascular disease, arthritis, andthe like.

Therefore, a pharmacological agent that increases CBF for a short periodof time would be of great benefit, particularly one that did not causeperipheral vasodilation. Vasodilators, for example dipyridamole, havebeen used for this purpose in patients prior to imaging withradionuclide. Dipyridamole is an effective vasodilator, but side effectssuch as pain and nausea limit the usefulness of treatment with thiscompound.

Adenosine, a naturally occurring nucleoside, also is useful as avasodilator. Adenosine exerts its biological effects by interacting witha family of adenosine receptors characterized as subtypes A₁, A_(2A),A_(2B), and A₃. AdenoScan® (Fujisawa Healthcare Inc.) is a formulationof a naturally occurring adenosine. AdenoScan has been marketed as anadjuvant in perfusion studies using radioactive thallium-201. However,its use is limited due to side effects such as flushing, chestdiscomfort, the urge to breathe deeply, headache, throat, neck, and jawpain. These adverse effects of adenosine are due to the activation ofother adenosine receptor subtypes in addition to A_(2A), which mediatesthe vasodilatory effects of adenosine. Additionally, the short half-lifeof adenosine necessitates multiple treatments during the procedure,further limiting its use. AdenoScan is contraindicated in many patientsincluding those with second- or third-degree block, sinus node disease,bronchoconstructive or bronchospastic lung disease, and in patients withknown hypersensitivity to the drug.

Other potent and selective agonists for the A_(2A) adenosine receptorare known. For example, MRE-0470 (Medco) is an adenosine A_(2A) receptoragonist that is a potent and selective derivative of adenosine. WRC-0470(Medco) is an adenosine A_(2A) agonist used as an adjuvant in imaging.These compounds, which have a high affinity for the A_(2A) receptor,and, consequently, a long duration of action, which is undesirable inimaging.

Thus, there is still a need for a method of producing rapid and maximalcoronary vasodilation in mammals without causing correspondingperipheral vasodilation, which would be useful for myocardial imagingwith radionuclide agents. Preferred compounds would be selective for theA_(2A) adenosine receptor and have a short duration of action (althoughlonger acting than compounds such as adenosine), thus obviating the needfor multiple dosing.

Selective A_(2A) receptor agonists are well known; for example, seeProvisional Patent Applications Ser. Nos. 60/1844296 and 60/21987. Thecompounds disclosed therein have a high specificity for the adenosineA_(2A) receptor subtype but are not necessarily selective to heart. Wehave discovered a method for identifying A_(2A) receptor agonists thatproduce the desired vasodilation in the heart but do not significantlyaffect the peripheral vasculature and have a short duration of action.

In addition to discovering a method to identify A_(2a) agonists that areselective coronary vasodilators, we have discovered that compounds thatmeet our criteria that would be superior as adjuncts to MPI techniques.

Additionally, in the above-identified Provisional Patent ApplicationsSer. Nos. 60/1,844,296 and 60/219,876 the effective dose of thecompounds of is disclosed to be in the range of 0.01-100 mg/kg/day.Surprisingly, we have discovered that the compounds are active at muchlower doses (0.0002-0.009 mg/kg) than those disclosed as effective inAccordingly, a novel and effective method of using the compounds isprovided, which is virtually free of undesirable side effects.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a method for identifyingcompounds useful as adjuncts in MPI, comprising the steps;

-   -   a. measuring the intrinsic efficacy of test compounds in a cell        line that stabily express adenosine A_(2A) receptors.    -   b. measuring the intrinisic efficacy of a full agonist in said        cell line.    -   c. selecting those compounds that have a lower intrinsic        efficacy than said full agonist;    -   d. measuring the binding affinity (K_(i)) of the selected        compounds; and    -   e. selecting a compound with a K_(i)>1 uM.

Such compounds are selective partial A_(2A)-adenosine receptor agonists,which produce coronary dilation in mammals without causing correspondingperipheral vasodilation at significant levels. They are low affinitycompounds having a short duration of action.

In a second aspect, the invention relates to a method of measuringcoronary blood flow (CBF) in a mammal, comprising administering to amammal low doses of an A_(2A) agonist referred to as CVT-3033, orCVT-3146.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Competititive radiolabeling binding assays of adenosine receptoragonists for A_(2A) and A₁ binding sites. Membranes prepared fromHEK-293 expressing human A_(2A) adenosine receptors were incubated with[³H]ZM241385 (1.5-5 M) and from 10⁻⁹M-10⁻⁵ M of the various agonists(FIG. 1A). Membranes from CHO-K1 cells expressing human A₁ adenosinereceptors were incubated with [³H]CPX (2.5-3.0 M) and from 10⁻⁹M-10⁻⁵ Mof the various agonists (FIG. 1B). The cells were incubated for twohours at room temperature in 50 mmol/L Tris-HCl buffer (pH 7.4)containing ADA (1 U/mL) and 100 μM Gpp(NH)p. Non-specific binding of [³H]ZM241385 or [³H]CPX was determined in the presence of either 100μmol/L NECA or 1 μmol/L CPX, respectively. Each point represents themean±SEM of triplicates pooled from at least three experiments. Valuesof K_(i) and pK_(i) are given in Table 2.

FIG. 2. Competititive radiolabeling binding assays of adenosine receptoragonists for A_(2b) and A₃ binding site. Membranes suspensions fromHEK-293 cells expressing A_(2b) adenosine receptors were incubated with[³ H]DPCPX (1.5-5 M) and from 10⁻⁸M-10⁻⁵ M of the various agonists (FIG.2A). Membranes from CHO-K1 cells expressing A₃ adenosine receptors wereincubated with [¹²⁵I]ABMECA (2.5-3.0 M) and from 10⁻⁸M-10⁻⁵ M of thevarious agonists (FIG. 2B). The cells were incubated for two hours atroom temperature in 50 mmol/L Tris-HCl buffer (pH 7.4) containing ADA (1U/mL). Each point represents the mean±SEM of triplicates pooled fromthree experiments.

FIG. 3. Effects of adenosine receptor agonists on cAMP content in intactPC12 cells. (FIG. 3A) PC12 cells were incubated for 10 minutes withvarious concentrations of adenosine receptor agonists in the presence of50 μmol/L rolipram. Cyclic AMP levels were determined as described under“Methods”. Values represent mean±SEM of results of triplicate samplesfrom three experiments. (FIG. 3B) Effect of CVT-3033 (1 μM) on CGS21680stimulated increase cAMP accumulation in PC12 cells. PC12 cells werestimulated for 10 minutes with various concentrations of CGS21680 in theabsence or presence of the partial agonist CVT-3033 (1 μM). Valuesrepresent mean±SEM of triplicate determinants from one representativeexperiment.

FIG. 4. Effect of CVT-3146 and CVT-3033 on cAMP content in HEK-293cells. HEK-293cells were incubated for 10 minutes with variousconcentrations of CVT-3146 or CVT-3033 in the presence of 50 μmol/Lrolipram. Cyclic AMP levels were determined as described under“Methods”. Values represent mean±SEM of triplicate samples from threeexperiments.

FIG. 5. Time course of the decline in agonist stimulated cAMP followingaddition of an A_(2A) adenosine receptor antagonist SCH58261. (FIG. 5A)PC12 cells were stimulated with various adenosine receptor agonists inthe presence of 50 μmol/L rolipram at 37° C. After a 10 minuteincubation, SCH58261 (20 μmol/L) was added and cAMP content wasdetermined at the times indicated. (FIG. 5B) Linear regression analysisof the relationship between the T_(1/2) (min) and pK_(i) for theagonists. Binding affinities were determined by measurement of thedisplacement of specific binding of [³H] ZM241385 from membranesprepared from PC12 cells using the data from Table 2

FIG. 6. Effects of adenosine receptor agonists on coronary conductancein isolated rat hearts. Concentration-dependent stimulation by CGS21680,CVT-2995, WRC0470, CVT-3146, CVT-3033, CVT-3032 and CVT-3100 of coronaryconductance of isolated rat hearts. Symbols and error bars represent themean±SEM of single determination from 4 to 6 hearts per agonist. Heartswere paced at a cycle length of 250 msec. Coronary conductance in theabsence of drug was 0.17:0.01 ml/min/mmHg (mean±SEM, n=26).

FIG. 7A. Effect of CPX and ZM241385 on CVT-3146 (10 nM) on CPP byisolated rat hearts. FIG. 7B. Effect of concentration of ZM241385 onCVT-3146 stimulation of CC by isolated rat hearts.

FIG. 8. Functional selectivity of CVT-3146 and CVT-3033 for adenosinereceptor subtypes. (FIG. 8A) Effect of concentration on AV conductiontime and coronary conductance in isolated rat hearts. Symbols and errorbars represent means±SEM of single determinations from each of threehearts. (FIG. 8B)

FIG. 9. Effect of adenosine receptor agonists on coronary perfusionpressure (CCP) in isolated rat hearts. Decreases in CPP caused byinfusion of CVT-3146 (10 nM, 4 min) or CGS21680 (100 nM, 4 min) (FIG. 9Aand FIG. 9B). Decrease in CPP caused by the infusion of CVT-3146 (10 nM)with or without additional infusion of CGS21680 (100 nM).

FIG. 10. Reversal of effect of agonist stimulation on CC. CVT-3033,CGS21680 and adenosine were given as boluses by iv infusion to isolatedrat hearts and then. CC was measured at 8, 16 and 24 minutes afteradministration (FIG. 10A). Also, CVT-3146, WRC 0470 and adenosine weregiven as boluses by iv infusion to isolated rat hearts and then CC wasmeasured at 8, 16 and 24 minutes after administration (FIG. 10B). Linearregression analysis of the relationship between the pK_(i) (data fromTable 2) and the reversal time (t_(0.9)) of coronary vasodilation aregiven in FIGS. 10C and D. Each data point represents the mean±SEM ofpK_(i) and T_(0.9) values. R and N are the correlation coefficient andnumber of agonists, respectively.

FIG. 11. Increases in CBF caused by CVT-3146 and adenosine in consciousdogs. Each data point is mean±SEM of the peak effect in CBF from 6 dogs.*: p<0.05, †: p<0.01 and ‡: p<0.001. Adenosine: (O) and CVT-3146 (O).

FIG. 12. Time course of changes in average peak coronary (O) andperipheral (O) artery blood flow velocity after an IV bolus injection ofCVT-3146 (1 ug/kg) (A) and adenosine (200-300 μg/kg).(B). Each pointrepresents the changes in average peak flow velocity (ΔAPV) incomparison with the baseline values and represent the mean±SEM of singledeterminations.

DETAILED DESCRIPTION

Definitions and General Parameters

This invention provides methods for identifying partial adenosine A_(2A)receptor agonists, which are particularly useful in MPI.

The interaction of an adenosine A_(2A) agonist with the extracellulardomain of its receptor causes a cascade of intracellular responses thatculminate with vasodilation. However, there are quantitative differencesin the ability of various agonists to achieve this effect. With respectto drug design, the ideal agent for imaging would have a duration ofaction that is brief enough not to cause serious side effects, but longenough to obviate the necessity for multiple treatments. It would beselective for the A_(2A) receptor, thus avoiding side effects due tointeraction with other adenosine subtypes, and it would produce coronaryvasodilation without causing corresponding peripheral vasodilation.

Compounds that act as A_(2A) agonists produce a variety of effects thatdepend on both the characteristics of the agonist, its receptor, and thetissue bearing A_(2A) receptors. Factors relate to agonist propertiesare the intrinsic efficacy (E) and the equilibrium dissociation constantof the agonist-receptor complex (K_(d)).

Intrinsic efficacy (maximal efficacy) is the maximum effect that anagonist can produce if the dose is taken to its maximum. Efficacy isdetermined mainly by the nature of the receptor and its associatedeffector system. By definition, partial agonists have a lower maximalefficacy than full agonists.

The K_(d) of a drug is obtained from data generated from a saturationexperiment analyzed according to a Scatchard plot (B/F versus F), whichleads to a linear curve. The K_(d) is estimated as the negativereciprocal of the slope of the line of best fit, and B_(max) by theabscissa intercept of the line. The reciprocal of K_(d) measures theaffinity constant (K_(a)) of the radioligand for the receptor. Thus, fora given ligand-receptor pair, the smaller the K_(d) (0.1-10 nM) thehigher its affinity. B_(max) is expressed as pmol or fmol per mg tissueor protein.

When the saturation experiment is performed in the presence of adisplacer (competitor), the line of best fit of the Scatchard plot canbe modified in a manner that depends on the type of receptor interactionexhibited by the displacer. Two main cases exist: (1) decreased slopeand unchanged B_(max) the displacement is of the competitive type; (2)unchanged slope and unchanged displacement of the non-competitive type.Intermediate cases where both the slope and B_(max) are modified alsoexist.

Data generated from a displacement experiment are generally fitted by asigmoidal curve termed the displacement or inhibition curve, that is thepercentage radiolabeled ligand specifically bound versus log [displacer]in M). The abscissa of the inflexion point of the curve gives the IC₅₀value, the concentration of displacer that displaces or inhibitor 50% ofthe radioactive ligand specifically bound. IC₅₀ is a measure of theinhibitor or affinity constant (Ki) of the displacer for the receptor.IC₅₀ and K_(i) are linked as follows if the displacement is of thecompetitive type thenK _(i)=IC₅₀/(1+[C*]/K _(d)*

This is the Cheng-Prusoff equation (Biochem. Pharmacol, 22:3099 (1973)).[C*] is the concentration of radioligand and K_(d)* is its dissociationconstant. The duration of the biological effect of an agonist isdirectly related to the binding affinity of a compound. It is desirablethat compounds that act as adjuncts in imaging have an effect that islong enough to produce a response without repeated administration butshort enough to avoid adverse side effects. Consequently, the preferredcompounds of the invention will have a relatively low binding affinityand a relatively short duration of action.

The potency is the dose or concentration required to bring about somefraction of a compound's maximal effect (i.e., the amount of compoundneeded to produce a given effect). In graded dose-response measurements,the effect usually chosen is 50% of the maximum effect and the dosecausing the effect is called the EC₅₀. Dose-response ratios using EC₅₀values for an agonist for a given receptor in the absence and presenceof various concentrations of an antagonist for the same receptor aredetermined and used to construct a Schild plot from which the K_(b) and_(p)A₂ (−log₁₀K_(b)) values are determined.

The concentration of antagonist that causes 50% inhibition is known asthe IC₅₀. IC₅₀ is used to determine the K_(b), theequilibriumdissociation constant for the antagonist-receptor complex.Thus,K _(b)=[IC₅₀]/1+[A]/K _(A)Wherein K_(A)=equilibrium dissociation constant for an agonist bindingto a receptor (concentration of agonist that causes occupancy of 50% ofthe receptors) and [A] is the concentration of agonist.

A compound may be potent but have less intrinsic activity than anothercompound. Relatively potent therapeutic compounds are preferable to weakones in that lower concentrations produce the desired effect whilecircumventing the effect of concentration dependent side effects.

The tissue specific factors that determine the effect of an agonist arethe number of viable specific receptors in a particular tissue [RT] andthe efficiency of the mechanisms that convert a stimulus (S) into aneffector response. Thus, there exists for a given tissue, a complexfunction f (S) that determines the magnitude of the response:

${Response} = {{f(S)} = \frac{f\left( {\lbrack A\rbrack{E\lbrack{RT}\rbrack}} \right)}{\left( {\lbrack A\rbrack + K_{d}} \right)}}$Therefore, a response to a drug is a function of both the stimulusproduced by agonist interaction with the receptor and the efficiency ofthe transduction of that stimulus by the tissue. Stimulus isproportional to the intrinsic efficacy of the agonist and the number ofreceptors. Consequently, variation in receptor density in differenttissues can affect the stimulus for response. Furthermore, some tissueshave very efficiently coupled receptors and other relatively inefficientcoupled receptors. This has been termed ‘receptor reserve’ (or sparereceptor) since in the first case, a maximum effect can be achieved whena relatively small fraction of the receptor is apparently occupied andfurther receptor occupancy can produce no additional effect. Themagnitude of the response will thus depend on the intrinsic efficacyvalue so that, by classical definition, full agonists (E=1) produce themaximum response for a given tissue, partial agonists produce a maximumresponse that is below that induced by the full agonist (0≦E≦1), andantagonists produce no visible response and block the effect of agonists(E=0). These activities can be completely dependent upon the tissue,i.e., upon the efficiency coupling. Therefore, low-efficacy adenosineagonists may be partial agonists in a given tissue and yet full agonistsin peripheral arteries with respect to a function such as vasodilation.

The presence of spare receptors in a tissue increases sensitivity to anagonist. An important biologic consequence of spare receptors is thatthey allow agonists with low efficacy for receptors to produce fullresponses at low concentrations and therefore elicit a selective tissueresponse. Thus, a drug may be designed to elicit a maximal effect in adesired tissue but elicit a less than maximal effect in other tissueswhen such action of a drug would lead to undesirable side effects.

Thus, the invention provides a method of identifying drugs by firstdetermining their efficacy compared to a known full agonist. Then, thebinding affinity of the compound is determined. Compounds identified bythis method will demonstrate partial agonist effects in the cAMP assaysand a low K_(i) as determined by affinity binding assays.

One preferred compound of the invention that is a selective partialA_(2A)-adenosine receptor agonist with a short duration of action is acompound of the formula:

CVT-3033 is particularly useful as an adjuvant in cardiological imaging.The preparation of CVT-3033 and related compounds is described in U.S.patent application Ser. No. 09/338185, filed on Jun. 22, 1999, thespecification of which is incorprated herein by reference.

Another preferred compound of the invention that is a selective partialA_(2A)-adenosine receptor agonist with a short duration of action is acompound of the formula:

The preparation of CVT-3146 and related compounds is set forth in U.S.patent application Ser. No. 09/338327 filed on Jun. 22, 1999, thespecification of which is incorporated herein by reference.

Compounds identified by the method of the invention are partial A_(2A)agonists that increase CBF but do not significantly increase peripheralblood flow. That is, the stimulation of blood flow in the periphery isless than 50% of the increase of that in the heart.

Preferred compounds identified by the method of the invention have aduration of less than 5 seconds but longer than the effect produced byadenosine.

The compounds identified by the method of the invention are usefulA_(2A) agonists that may be used as adjuncts in cardiac imaging whenadded either prior to dosing with an imaging agent or simultaneouslywith an imaging agent.

Suitable imaging agents are ²⁰¹Thallium or ^(99m)Technetium-Sestamibi,^(99mTc)teboroxime, and ^(99mtc)(III).

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances in which said event or circumstanceoccurs and instances in which it does not.

The compositions may be administered orally, intravenously, through theepidermis or by any other means known in the art for administeringtherapeutic agents.

The method of treatment of comprises the administration of an effectivequantity of the chosen compound, preferably dispersed in apharmaceutical carrier. Dosage units of the active ingredient aregenerally selected from the range of 0.3 to 103 μg/kg, but will bereadily determined by one skilled in the art depending upon the route ofadministration, age and condition of the patient. These dosage units maybe administered one to ten times daily for acute or chronic disorders.No unacceptable toxicological effects are expected when compounds of theinvention are administered in accordance with the present invention.

Pharmaceutical compositions including the compounds of this invention,and/or derivatives thereof, may be formulated as solutions orlyophilized powders for parenteral administration. Powders may bereconstituted by addition of a suitable diluent or otherpharmaceutically acceptable carrier prior to use. If used in liquid formthe compositions of this invention are preferably incorporated into abuffered, isotonic, aqueous solution. Examples of suitable diluents arenormal isotonic saline solution, standard 5% dextrose in water andbuffered sodium or ammonium acetate solution. Such liquid formulationsare suitable for parenteral administration, but may also be used fororal administration. It may be desirable to add excipients such aspolyvinylpyrrolidinone, gelatin, hydroxy cellulose, acacia, polyethyleneglycol, mannitol, sodium chloride, sodium citrate or any other excipientknown to one of skill in the art to pharmaceutical compositionsincluding compounds of this invention. Alternatively, the pharmaceuticalcompounds may be encapsulated, tableted or prepared in an emulsion orsyrup for oral administration. Pharmaceutically acceptable solid orliquid carriers may be added to enhance or stabilize the composition, orto facilitate preparation of the composition. Liquid carriers includesyrup, peanut oil, olive oil, glycerin, saline, alcohols and water.Solid carriers include starch, lactose, calcium sulfate, dihydrate,teffa alba, magnesium stearate or stearic acid, talc, pectin, acacia,agar or gelatin. The carrier may also include a sustained releasematerial such as glycerol monostearate or glycerol distearate, alone orwith a wax. The amount of solid carrier varies but, preferably, will bebetween about 20 mg to about 1 gram per dosage unit. The pharmaceuticaldosages are made using conventional techniques such as milling, mixing,granulation, and compressing, when necessary, for tablet forms; ormilling, mixing and filling for hard gelatin capsule forms. When aliquid carrier is used, the preparation will be in the form of a syrup,elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquidformulation may be administered directly or filled into a soft gelatincapsule.

The Examples that follow serve to illustrate this invention. TheExamples are intended to in no way limit the scope of this invention,but are provided to show how to make and use the compounds of thisinvention.

EXAMPLES

The following abbreviations are used in the Examples:

TABLE 1 List of Abbreviations and Activities of Experimental CompoundsChemical Compound Abbreviation Receptor/Activity4-aminobenzyl-5′-N-methylcarbox- ABMECA A₃ agonist amidoadenosineadenosine deaminase ADA 2-p-(2-carboxy-ethyl) phenethyl- CGS21680: Highaffinity amino-5′-N-ethylcarboxamido- A_(2A) agonist adenosine8-cyclopentyl-1,3-dipropylxanthine CPX A₁ antagonist5′-guanylyl-imididodiphosphate Gpp(NH)p Stabilizes GPCR2-hexynyladenosine-5′-N-ethyl- HENECA N-ethylcarboxamido-adenosine NECANon-selective adenosine receptor agonist Phenylisopropyladenosine R-PIAA₁ receptor agonist Rolipram phosphodiesterase inhibitor SCH58261:A_(2A) antagonist 2-cyclohexylmethylidenehydra- WRC-0470: High affinityzinoadenosine 2A agonist 4-(2-[7-amino-2-(2furyl)[1,2,4]- ZM241385: 2Aantagonist triazolo[2,33-a][1,3,5]triazin-5-yl amino]ethyl)phenol

Other abbreviations include cAMP (cyclic adenosine monophosphate), APV(average peak velocity), CBF (coronary blood flow), CHO-Ki (Chinesehamster ovary cell line), HEK-293 (human cell line), CPP (coronaryperfusion pressure), CR (coronary resistance), HR (heart rate), im(intramuscular), iv (intravenous) LVSP (left ventricle systolicpressure), MAP (mean arterial pressure). PBF (peripheral blood flow).

Adenosine deaminase was purchased from Boehringer Mannheim BiochemicalsIndianapolis, Ind.). [³H] ZM241385 was purchased from Tocris Cookson Ltd(Langford, Bristol, UK). [³H] CPX was from New England Nuclear (Boston,Mass.). HENECA CGS21680, adenosine, NECA, R-PIA, phenylephrine, DMSO,rolipram and HEK-hA_(2A)AR membranes were obtained from Sigma-RBI(Natick, Mass.). Nitroglycerin was obtained from Parke-Davis, MorrisPlains, N.J. Aminophylline was obtained from Abbott Laboratories,Chicago, Ill.

HENECA was a gift from Professor Gloria Cristalli of the University ofCamerino, Italy. Drug stock solutions (10 mmol/L) were prepared in DMSO.Sprague Dawley rats were purchased from Simonsen Laboratories (Gilroy,Calif.). Ketamine was purchased from Fort Dodge Animal Health (FortDodge, Iowa) and xylazine from Bayer (Shawnee Mission, Kans.). SuccinylcAMP-tyrosyl methyl ester (ScAMP-TME) was purchased from Sigma andiodinated in the presence of chloramine T. CVT-2995,CVT-3003-((4S,2R,3R,5R)-2-(6-amino-2-(2-thienyl)pruin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol),CVT-3006-((4S,2R,3R,5R)-2-(6-amino-2-{3-[2-benzylphenoxy]prop-1-ynyl}purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol),CVT-3032, CVT-3033,CVT-3100-((4S,2R,3R,5R)-2-[6-amino-2-(5-methyl(2-thienyl))purin-9-yl]-5-hydroxymethyl)oxolane-3,4-dion),CVT-3101-(4-(3-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}prop-2-ynyloxy)benzenecarbonitrile),CVT-3126-(4S,2R,3R,5R)-2-{6-amino-2-[1-(3-phenylpropyl)pyrazol-4-yl]purin-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol),CVT-3127-(ethyl1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydromethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylate),CVT-3141-((4S,2R,3R,5R)-2-{6-amino-2-[4-(4-chlorophenyl)pyrazole]purin-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol),CVT-3144-((4S,2R,3R,5R)-2-{6-amino-2-[4-(4-methylphenyl)pyrazolyl]purin-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol),CVT-3146, YT-146 and WRC0470 were synthesized by CV Therapeutics,Department of Medicinal and Bioorganic Chemistry, Palo Alto, Calif. Thestructures of several of these compounds are set forth on the followingpage.

Examples 1 and 2

Examples 1 and 2 demonstrate the selectivity and binding affinity of thecompounds of the invention to adenosine receptors on cells obtained asfollows. Rat pheochromocytoma PC12 cells were obtained from the AmericanType Culture Collection and grown in DMEM with 5% fetal bovine serum,10% horse serum, 0.5 mmol/L L-glutamine, 100 U/mL penicillin, 0.1 mg/mLstreptomycin, and 2.5 μg/mL amphotericin.

HEK-293 cells stabily expressing recombinant human A_(2B) adenosinereceptors (HEK-hA_(2B) adenosine receptors) were grown in DMEMsupplemented with 10% fetal bovine serum and 0.5 mg/mL G-418.

CHO-K1 cells stabily expressing the recombinant human A₁ adenosinereceptors (CHO-hA₁ adenosine receptors) or A₃ adenosine receptors(CHO-hA₃ adenosine receptors) were grown as monolayers on 150-mm plasticculture dishes in Ham's F-12 media supplemented with 10% fetal bovineserum in the presence of 0.5 mg/mL G-418. Cells were cultured in anatmosphere of 5% CO₂/95% air and maintained at 37° C.

Cell membranes were harvested from the cell lines by detaching cellsfrom the culture plates into ice-cold 50 mmol/L Tris-HCl buffer (pH7.4). The cell suspensions were homogenized and centrifuged at 48,000 gfor 15 minutes. The pellets were washed three times by re-suspension inice-cold Tris-HCl buffer and centrifugation. The final pellet wasre-suspended in Tris-HCl, aliquoted and frozen at −80 C. until used forreceptor binding assays. The protein concentration of membranesuspensions was determined using the Bradford (Bradford, M. M. (1976.Anal. Biochem. 72, 248) with bovine serum albumin as standard.

Membranes were also obtained from porcine striatial cells as follows.Porcine striatum was obtained from Pel Freeze Inc. Striatum was mincedand homogenized in 10 volumes of ice-cold 50 mmol/L Tris HCl buffer(pH7.4). The homogenate was filtered through cotton gauze andcentrifuged at 48,000 g for 15 minutes at 4° C. The supernatant wasdiscarded, and the membrane pellet was suspended in 10 volumes of 50mmol/L Tris-HCl buffer (pH 7.4) and washed three times by centrifugationand resuspended in fresh buffer. The final pellet was frozen at −80° C.until used for receptor binding assays.

Competitive radiolabeling binding assays were performed to determine thebinding affinities of the compounds of the invention for the adenosinereceptor subtypes, A₁, A_(2A), A_(2B) and A₃. Briefly, membranesuspensions, obtained from cells expressing either adenosine receptorsubtypes A₁, A_(2A), A_(2B) or A₃ were incubated for 2 hours at roomtemperature in 50 mmol/L Tris-HCl buffer (pH 7.4) containing ADA (1U/mL). [³H]-ZM241385 (˜1.5 to 5 nmol/L) was added to membranes fromcells expressing A_(2A), [³H]-CPX (˜2.5 to 3.0 nmol/L) was added tomembranes from cells expressing A₁, [³]-CPX (30 nM) was added tomembranes from cells expressing A_(2B) and [¹²⁵I]ABMECA (1 nM)} wasadded to membranes from cells expressing A₃. The competing agents, thatis the agonists (10⁻⁹-10⁻⁴M) were also added along Gpp (NH) p (100 μg)which stabilizes the receptor in the low affinity state therebyobviating the complication of multiple affinity states (Gao., Z. et al.(1999). Biochem J 338(3):729). At the end of the incubation, freeradioligand was separated from membrane-bound radioligand by filtrationthrough Whatman GF/C glass fiber filters using a Brandel tissueharvester (Gaithersburg, Md.). Triplicate determinations were performedfor each concentration of unlabelled compound.

The results of radioligand binding assays, presented in Table 2, showthat binding affinities of CVT-3033 (1417 nM) and CVT-3146 (1095 nM) toA_(2A) receptors in pig striatum were lower (i.e. K_(i) higher) than afull A_(2A) agonist, CGS21680 (157 nM).

The binding affinities of CVT-3033 (3623 nM) and CVT-3146 (1734 nM) toA_(2A) were also lower than CGS21680 (210 nM) in PC12 cells. Bindingaffinities of CVT-3033 (2895 nM) and CVT-3146 (1269 nM) were also lowerthan CGS21680 (609 nM in HEK cells expressing human A_(2A) receptors.CGS-21680, CVT-3033, and CVT-3146 had relatively low affinities for theA₁ receptor expressed by CHO-K1 cells. Binding to the A_(2A) receptorwas relatively greater than binding affinities to A₁, A_(2b) or A₃(FIGS. 1A and B and FIGS. 2A and B).

TABLE 2 Binding affinity of adenosine receptor agonists for A_(2A) andA₁ AdoRs K_(i9) nmol/L pK₁ ± SEM Pig Striatum (A_(2A)) PC12 cells(A_(2A)) HEK-hA_(2A)AdoR CHO-hA₁AdoR Compounds Affinity N Affinity NAffinity N Affinity N NECA ND 297 (6.54 ± 0.86) 4 360 (6.45 ± 0.06) 3328 (6.49 ± 0.06) 3 R-PIA 1106 (5.96 ± 0.06) 4 3476 (5.48 ± 0.10) 3 1656(5.78 ± 0.02) 3 477 (6.45 ± 0.11) 3 CGS21680 157 (6.85 ± 0.23) 5 210(6.74 ± 0.09) 3 609 (6.22 ± 0.06) 3 3540 (5.47 ± 0.20) 3 CVT-2995 14.4(7.86 ± 0.12) 4 53 (7.20 ± 0.05) 4 305 (6.52 ± 0.04) 6 866 (6.07 ± 0.05)3 WRC0470 20.7 (7.68 ± 0.04) 3 18 (7.77 ± 0.04) 8 272 (6.55 ± 0.04) 67278 (5.16 ± 0.09) 3 CVT-3032 1594 (5.80 ± 0.10) 3 5516 (5.26 ± 0.05) 313651 (4.87 ± 0.02) 3 6350 (5.22 ± 0.11) 3 CVT-3033 1417 (5.67 ± 0.16) 33623 (5.45 ± 0.03) 5 2895 (5.54 ± 0.03) 3 5836 (5.24 ± 0.04) 3 CVT-31461095 (5.95 ± 0.11) 5 1734 (5.76 ± 0.01) 3 1269 (5.90 ± 0.03) 7 >16460(4.59 ± 0.35) 3 YT-146 16.3 (7.86 ± 0.33) 4 ND ND ND CVT-3003 1007 (6.00± 0.07) 3 ND ND ND CVT-3006 64 (7.24 ± 0.26) 3 ND ND ND CVT-3100 697(6.16 ± 0.06) 5 ND ND ND CVT-3101 94 (7.03 ± 0.06) 3 ND ND ND CVT-31261667 (5.78 ± 0.09) 3 ND ND ND CVT-3127 64 (7.22 ± 0.17) 3 ND ND NDCVT-3141 1138 (5.95 ± 0.07) 3 ND ND ND CVT-3144 502 (6.31 ± 0.12) 3 NDND ND HENECA 7.96 (8.10 ± 0.08) 10 ND ND ND The binding affinities ofadenosine receptor agonists for A_(2A)AdoRs and A₁AdoRs were determinedby their effect to compete with specific binding of [³H]ZM241385 or[³H]CPX, respectively, to membranes from the indicated tissue/cells.Values are mean ± SEM of results of at least three experiments (n)performed in triplicate. Numbers in parentheses are means of pK_(i) ±SEM. Values of the equilibrium dissociation constant(K_(d)) for[³H]ZM241385 binding that were used in calculations of K_(i) values were0.5, 0.5 and 0.8 nM for pig striatum, PC12 and HEK-hA_(2A)AdoR cells,respectively. A K_(d) value of 1 nM for [³H]CPX binding to CHO-hA₁AdoRmembrane was used in calculation of K_(i) values is 1.

Example 3

Example 3 demonstrates the ability of the compounds of the invention tostimulate cAMP levels, a measure of the intrinsic efficacy of theagonists. Briefly, PC12 cells were rinsed three times with Hanks'balanced saline solution (HBSS), detached using a cell lifter, andpelleted by centrifugation at 500 g for 5 minutes. Aliquots of the cellsuspension (0.1 to 0.2 mg protein) were placed in microfuge tubes with250 μL of HBSS containing rolipram (50 μmol/L) to inhibitphosphodiesterases that degrade cAMP_and warmed to 37° C. Appropriatedrugs were added to the cell suspensions, and incubations were allowedto continue for 10 minutes. Tubes were placed in a boiling water bathfor 5 minutes to terminate the incubation. The samples were then cooledto room temperature, diluted by the addition of 1 mL of 10 mmol/LTris-HCl buffer at pH 7.4, and then centrifuged for 2 minutes at 13000g.

The cAMP content of the supernatant was determined by modification of aradioimmunoassay method described by Harper and Brooker (1975. J. Cyclicnucleotide Res 1:207). Briefly, an aliquot of the supernatant (0.01 mL)was mixed with 0.04 mL of HBSS, 0.05 mL of 50 mmol/L sodium acetatebuffer (pH 6.2) containing 10 mmol/L CaCl₂, [¹²⁵I]ScAMP-TME (12500 dpm),and 0.05 mL of anti-cAMP antibody (1:2000 dilution with 0.1% bovineserum albumin in distilled water). The samples were then incubated at 4°C. for 16 hours. At the end of the incubation, 70 μL of a 50% (wt/vol)hydroxyapatite suspension was added to each tube. The suspensions weregently agitated and then incubated for 10 minutes at 4° C.Antibody-bound radioactivity adsorbed to hydroxyapatite was collectedonto glass fiber filters by vacuum filtration using a Brandel cellharvester. Radioactivity retained by the filter was counted in a gammacounter. Nonspecific binding of [¹²⁵I]ScAMP-TME was defined asradioactivity bound in the presence of 3 μmol/L unlabeled cAMP and wassubtracted from total binding. The amount of cAMP present in samples wascalculated based on a standard curve using known amounts of cAMP.

As illustrated in FIG. 3A, all compounds increased the cellular contentof cAMP in a concentration-dependent manner. The low affinity A_(2A)agonists CVT-3032, CVT-3033 and CVT-3146, were not only less potent(10-15 fold), but also less effective in stimulating cAMP accumulationcompared to CGS21680. The maximal responses induced by CVT-3146,CVT-3033 and CVT-3032 were 85%, 63% and 65% of that induced by CGS21680,respectively. These data demonstrate that CVT-3146, CVT-3033 andCVT-3032 behave as partial A_(2A) agonists in PC12 cells. Additionally,CVT-3033 (1 μM) inhibit the ability of CGS21680 to stimulate cAMP levels(FIG. 3B) causing an approximate 5-fold shift to the right of theCGS21680 concentration-response curve

It is notable that the stimulation of cAMP levels in PC12 cells wasrelated to the binding affinity of the compounds to A_(2A) receptors(Table 3).

TABLE 3 Rate of decline (t_(0.5)) of cAMP accumulated after exposure tothe A_(2A) agonists in PC12 cells Agonists t_(0.5) (min) n WRC0470 5.9 ±0.8 6 CGS21680 5.3 ± 0.5 5 CVT-2995 3.9 ± 0.6 5 CVT-3146 2.6 ± 0.2 6CVT-3033 1.9 ± 0.1 4 R-PIA 1.6 ± 0.2 6 Values are mean ± SEM of theleast three experiments performed in triplicate. The pK₁ (-logK₁) valueswere obtained from the results of competition binding assays under[³H]ZM241385 as the radioligand for A_(2A) AdoRs. The pEC50 (-logEC50)values were determined from concentration response relationships foragonist-induced cAMP accumulation in PC12 cells. The t_(0.5) values werecalculated from the rate of decline of cAMP accumulated during exposureto each agonist (1 μM) following the addition of the A_(2A) AdoRantagonists SCH58261 (20 μM)>

The selectivity of the effects of CVT-3033 and CVT-3146 on cAMPaccumulation in HEK-293 cells expressing A_(2B) adenosine receptors isalso shown. NECA, a non-selective adenosine receptors agonist, caused aconcentration-dependent increase of cellular cAMP content whereasneither CVT-3033 nor CVT-3146 had any detectable effects even at a highconcentration of 100 μM (FIG. 4). These results indicate that CVT-3033and CVT-3146 have very weak, if any, interaction with A_(2B) receptors.

The effect of an A₂a antagonist on agonist-mediated cAMP accumulation inPC12 was also demonstrated. PC12 cells cultured in DMEM at 37° C. weretreated with WRC0470, CGS21680, CVT-2995, CVT-3146, CVT-3033 and R-PIAeach at a concentration of 1 μM in the presence of rolipram (50 μM) for10 minutes. Then, an A_(2A) antagonist, SCH58261 (20 μM), was added andcAMP content was determined at various periods. The time for cAMP levelsto decrease to half maximal (t_(0.5)) was calculated and plotted againstthe affinity (pK_(i)) of each agonist for the A_(2A) adenosine receptor,as determined by competition radioligand binding assays (above).

FIG. 5A shows the time-course of the decline of agonist-stimulated cAMPaccumulation following the addition of SCH58261when compared to thecontrol cultures (CGS2160 incubated without SCH58261). The calculatedvalues of t_(0.5) from these experiments are presented in Table 3. Theapparent t_(0.5) values of agonists were inversely related to theiraffinities for A_(2A) adenosine receptors, that is, the greater theagonist affinity, the lower the rate of decline of cAMP content uponapplication of the A_(2A) adenosine receptors antagonist SCH58261 (FIG.5A). As depicted in FIG. 5B, the relationship between the apparentt_(0.5) and pK_(i) for the agonists was best fit by linear regressionwith a correlation coefficient (r value) of 0.84.

Example 4

The effect of the compounds of the invention on coronary conductance(CC), an estimate of vasodilation, was demonstrated ex vivo usingperfused rat hearts. Briefly, rats of either sex weighing 230-260 gramswere anesthetized by intraperitoneal injection of a mixture of ketamine(100 mg/ml) and xylazine (20 mg/ml). The chest of each rat was openedand the heart removed, and rinsed in ice-cold modified Krebs-Henseleit(K-H) solution containing NaCl 117.9, KCl 4.5, CaCl₂ 2.5, MgSO₄ 1.18,KH₂PO₄ 1.18, pyruvate 2.0 mmo/L. The aorta was cannulated and the heartwas perfused at a flow rate of 10 ml/min with modified K-H solution. TheK-H solution (pH 7.4) was gassed continuously with 95% O₂ and 5% CO₂ andwarmed to 35±0.5° C. The heart was electrically paced at a fixed cyclelength of 240 ms (250 beats/min) using a bipolar electrode placed in theleft atrium. The electrical stimuli were generated by a Grass stimulator(Model S48, W. Warwick, R.I.) and delivered through a Stimuli IsolationUnit (Model SIU5, Astro-Med, Inc., NY) as square-wave pulses of 3-msecin duration and with an amplitude of at least twice the thresholdintensity.

As shown in FIG. 6A, adenosine, CGS21680, WRC0470, and the CVT compoundscaused concentration-dependent increases in coronary conductance.

The potencies (EC₅₀ values) of adenosine, CGS21680, WRC0470 and the CVTcompounds are summarized in Table 4. The low affinity agonist CVT-3146was found to be approximately 10-fold more potent than adenosine but10-fold less potent than the high affinity agonists CGS21680 and WRC0470with respect to increasing coronary conductance. The results show thatCVT 3146 was a potent agonist of coronary conductance in heart but onlya weak agonist in PC 12 cells (EC₅₀=291 nM).

TABLE 4 Potency of adenosine and A_(2A) Adenosine receptor agonists toincrease cAMP accumulation in PC12 cells and coronary conductance in ratisolated perfused heart EC₅₀ (pEC₅₀ ± SEM), nM cAMP AccumulationCoronary Conductance Agonist (PC12 cells) (Rat Isolated Heart) CGS2168018 (7.75 ± 0.03) N = 3 0.54 (9.27 ± 0.03) N = 3 CVT-2995 6.6 (8.82 ±0.25) N = 3 0.68 (9.17 ± 0.03) N = 5 CVT-3146 291 (6.54 ± 0.03) N = 36.40 (8.19 ± 0.04) N = 4 CVT-3032 613 (6.21 ± 0.02) N = 3 66.50 (7.18 ±0.07) N = 4 CVT-3033 487 (6.31 ± 0.01) N = 3 67.95 (7.19 ± 0.08) N = 4WRC0470 ND 0.62 (9.19 ± 0.6) N = 5 Adenosine ND 59.20 (7.24 ± 0.11) N =4 Values are the mean concentrations of agonists that caused 50%increase in cAMP accumulation or coronary conductance (EC₅₀ and pEC₅₀).ND; Not determined.

Coronary vasodilatory effect of CVT-3146 in the absence and presence ofadenosine receptor antagonists were also demonstrated. The identity ofthe adenosine receptor subtype (A₁ or A_(2A)) mediating the coronaryvasodilation was determined. Hearts (n=6) were exposed to CVT-3146 (10nM), and after the effect of this agonist reached steady-state, CPX (60nM), an A1 antagonist and then ZM241385 (60 nM), an A2a antagonist wereadded to the perfusate and the changes in CPP were recorded. As depictedin FIG. 7A, CVT-3146 significantly increased coronary conductance to0.22±0.01 ml mm Hg⁻¹min⁻¹ from a baseline value of 0.16±0.02 ml mm Hg⁻¹min⁻¹. This increase in coronary conductance caused by CVT-3146 was notaffected by 60 nM CPX but was completely reversed by 60 nM ZM241385.Furthermore, the inhibition by ZM241385 of an increase of coronaryconductance caused by CVT-3146 was concentration-dependent (FIG. 7B).

A₁ adenosine receptor-mediated depression of A-V nodal conduction timeby CVT-3033 and CVT-3146 (negative dromotropic effect) was measuredusing atrial and ventricular surface electrograms as described byJenkins and Belardinelli (Circ Res 63:97).

As shown in FIG. 8, CVT-3146 and CVT-3033 increased coronary conductancein a concentration-dependent manner, but did not prolong A-V nodalconduction time Coronary perfusion pressure (CPP) was measured using apressure transducer that was connected to the aortic cannula via aT-connector positioned approximately 3 cm above the heart. CPP wasmonitored throughout an experiment and recorded either on a chartrecorder (Gould Recorder 2200S, Valley View, Ohio) or a computerizedrecording system (PowerLab/4S, ADInstruments Pty Ltd, Australia). Onlyhearts with CPP ranging from 60 to 85 mmHg (in the absence of drugs)were used in the study. CC conductance (in ml/min/mmHg) was calculatedas the ratio between coronary perfusion rate (10 ml/min) and CPP.

As shown in FIG. 9A the extent of the decrease in coronary perfusionpressure (an index of the coronary vasodilation) caused by CVT-3146 wassimilar to that caused by a supramaximal concentration of CGS21680 (FIG.9B). Both 10 nM CVT-3146 and 100 nM CGS21680 decreased coronaryperfusion pressure by 23 mmHg. In addition, in the presence of 10 nMCVT-3146, CGS21680 (100 nM) did not cause a further decrease of thecoronary perfusion pressure (FIG. 9C). Thus, CVT-3146 is a full agonistwith respect to coronary artery conductance.

The relationship between the affinity of agonists for the A_(2A)receptor and the rate of reversal of agonist-mediated responses(coronary vasodilation in heart and cAMP accumulation in PC12 cells)upon termination of drug administration were determined. All compoundswere given as boluses into the perfusion line at their respectiveminimal concentrations that caused equally or near-equally maximalincreases in coronary conductance. Likewise, the onset and time to peakeffect (i.e. maximal coronary vasodilation) were similar for allagonists. Although adenosine and the various agonists caused equalmaximal increases in coronary conductance, the durations of theireffects were markedly different. The duration of the effect of adenosinewas the shortest followed by those of CVT-3033 and CVT-3146. The effectsof CGS21680 and WRC0470 had the longest duration (FIG. 10). The durationof the coronary vasodilation in isolated rat hearts caused by adenosine,the CVT compounds, and other agonists measured as the time to 50% and90% (t_(0.5) and t_(0.9), respectively) reversal of the increases incoronary conductance after termination of drug administration aresummarized in Table 4. The reversal time of coronary vasodilationcorrelated with the affinity of the adenosine derivatives for the A_(2A)receptors. As shown in FIG. 9C, there was a significant (p<0.05) inverserelationship (r=0.87) between the affinity (pK_(i)) of the agonists forthe A_(2A) adenosine receptors (Table 2) and the reversal time (t0.9)(Table 4) of the coronary vasodilation caused by the same agonists inrat isolated hearts.

Example 5

The magnitude of the effect of A_(2A) adenosine receptor agonists oncoronary dilation and the duration of the effect was determined in pigsweighing 22-27 kg. All animals received humane care according to theguidelines set forth in “The Principles of Laboratory Animal Care”formulated by the National Society for Medical research and the “Guidefor the Care and Use of Laboratory Animals” prepared by the Institute ofLaboratory Animal Resources and published by the National Institutes ofHealth (NIH Publication No. 86-23, revised 1996). In addition, animalswere used in accordance with the guidelines of the University ofKentucky Institutional Animal Care and Use Protocol.

Animals were anesthetized with ketanine (20 mg/kg, i.m.) and sodiumpentobarbital (15-18 mg/kg, i.v.). Anesthesia was maintained withadditional sodium pentobarbital (1.5-2 mg/kg, i.v.) every 15-20 minutes.Animals were ventilated via a tracheotomy tube using a mixture of roomair and 100% O₂. Tidal volume, respiratory rate and fraction of O₂ ininspired air were adjusted to maintain arterial blood gas (ABG) and pHvalues. Core body temperature was monitored with an esophagealtemperature probe and maintained at 37.0-37.5° C. by use of a heatingpad. Lactate Ringers solution was administered via an ear or femoralvein as an initial bolus of 300-400 ml followed by a continuous infusionat a rate of 5-7 ml/kg/hr. A catheter was inserted into the femoralartery to monitor arterial blood pressure.

The heart was exposed through a median sternotomy and suspended in apericardial cradle. Left ventricular pressure (LVP) was measured with a5F high fidelity pressure sensitive tip transducer (Millar Instruments,Houston, Tex.) placed in the left ventricular cavity via an apicalincision and secured with a purse string suture. A segment of the leftanterior descending coronary artery (LAD), proximal to the origin of thefirst diagonal branch, was dissected free of surrounding tissue. Atransit time perivascular flow probe (Transonic Systems Inc., Ithaca,N.Y.) was placed around this segment to measure CBF. Proximal to theflow probe, a 24-gauge modified angiocatheter was inserted forintracoronary infusions. All hemodynamic data were continuouslydisplayed on a computer monitor and fed through a 32-bit analog todigital converter into an online data acquisition computer withcustomized software (Augury, Coyote Bay Instruments, Manchester, N.H.).A_(2A) adenosine receptors agonists were dissolved in DMSO to producestock concentrations of 1-5 mM, which were diluted in 0.9% saline andinfused at rates of 1-1.5 ml/min via the catheter. The A_(2A) adenosinereceptors agonists were administered intracoronary.

Relationship between affinity of various agonists for A_(2A) adenosinereceptor and the reversal time of their effect to increase coronaryconductance was determined in pigs. Each experiment was preceded by a30-minute stabilization period following the completion of allinstrumentation of the animal. Baseline hemodynamic data were thenrecorded and an intracoronary infusion of an A_(2A) Adenosine receptorsagonist was initiated. Each infusion was maintained for 4-5 minutes toallow LAD CBF to reach a steady-state, after which the infusion wasterminated. The times to recovery of CBF by 50% (t_(0.5)) and 90%(t_(0.9)) of the difference from peak effect to baseline CBF wererecorded. Ten to 15 minutes after CBF returned to pre-drug values asecond infusion with a different agonist was started. In preliminarystudies it was found that the intracoronary infusion of adenosinereceptor agonists produced varying degrees of systemic hypotension, andhence, in all subsequent experiments, phenylephrine was administeredintravenously at dose of ˜1 μg/kg/min. Hemodynamic measurements weremade prior to and following the initiation of the phenylephrineinfusion. The phenylephrine infusion rate was adjusted during andfollowing the infusions of the adenosine receptor agonists to maintainarterial blood pressure within 5 mmHg of pre-infusion values. The-effectof a maximum of three different agonists was determined in eachexperiment.

All CVT-compounds as well as CGS21680 and other A_(2A) adenosinereceptors agonists (i.e., WRC-0470 and YT-146) caused significantincreases in CBF (Table 6). Selected doses of these compounds given ascontinuous (4 to 5 min) intracoronary infusions caused 3.1 to 3.8-foldincreases in CBF. Once it was established that all agonists causedcomparable increases of CBF (i.e., “fold increase”) and caused little orno changes in heart rate and mean arterial blood pressure (data notshown), the reversal time of their coronary vasodilatory effects wasdetermined. As summarized in Table 5 the reversal times of the effect ofthe low affinity, partial agonists, CVT-3146, CVT-3032 and CVT-3033,were shorter than those of CGS21680, WRC-0470 or YT-146. Moreimportantly, as depicted in FIG. 9D, there was a significant (p<0.05)inverse relationship (r=0.93) between the affinity (pK_(i)) of theA_(2A) Adenosine receptors agonists for pig brain striatum A_(2A)receptors and the reversal time (t_(0.9)) of coronary vasodilation inpig heart Table 2).

TABLE 5 Reversal time of coronary vasodilation by adenosine andadenosine receptor agonists in rate isolated perfused Agonist t_(0.5)(min) t_(0.9) (min) n R-PIA  7.9 ± 0.1 12.6 ± 0.8 3 CGS21680 14.5 ± 0.519.5 ± 09  3 CVT-2995 17.5 ± 1.2 23.2 ± 2.1 4 WRC0470 21.9 ± 0.9 27.9 ±1.4 6 CVT-3032  4.1 ± 0.3  9.8 ± 1.4 4 CVT-3033  3.4 ± 0.5  8.4 ± 2.2 4CVT-3146  5.2 ± 0.2 11.3 ± 1.1 5 YT-146 17.7 ± 1.0 25.8 ± 4.0 3 CVT-3003 3.4 ± 0.1  9.2 ± 2.2 4 CVT-3006 16.1 ± 0.1 21.8 ± 2.0 3 CVT-3100  5.1 ±0.6 10.1 ± 0.2 4 CVT-3101 16.7 ± 0.5 25.6 ± 0.3 3 CVT-3126  8.3 ± 0.412.6 ± 0.3 4 CVT-3127 14.8 ± 2.1 15.0 ± 0.8 3 CVT-3141 14.4 ± 1.9 21.3 ±3.9 4 CVT-3144 13.6 ± 1.3 18.9 ± 1.9 4 HENECA 28.6 ± 1.1 32.8 ± 3.1 3Adenosine  1.6 ± 0.1  5.6 ± 0.8 11 Times (in minutes) to 50% and 90%(t_(0.5) and t_(0.9), respectively) reversal of the increases incoronary conductance caused by adenosine and adenosine receptoragonists. Values are the mean ± SEM of single determination in each of npreparation.

TABLE 6 Magnitude and reversal time of coronary dilation caused byvarious adenosine receptor agonists in open-chest anesthetized pigs CBF(fold- Agonist Dose increase) t_(0.5) (min) t_(0.9) (min) n CVT-3146 10ug/kg/min 3.40 ± 0.04 1.9 ± 0.2 10.1 ± 0.7 3 CVT-3146 30 ug/kg/min 3.38± 0.39 2.6 ± 0.5 12.3 ± 1.1 6 CVT-3032 30 ug/kg/min 3.78 ± 0.70 2.3 ±0.6  9.6 ± 1.0 3 CVT-3033 50 ug/kg/min 3.33 ± 0.58 3.1 ± 0.9 12.0 ± 1.03 WRC0470  1 ug/kg/min 3.14 ± 0.24 9.5 ± 0.8 22.5 ± 1.6 6 CGS21680  2ug/kg/min 3.54 ± 0.09 9.7 ± 0.8 21.4 ± 0.8 3 YT-146  1 ug/kg/min 3.44 ±0.47 17.8 ± 3.4  32.9 ± 5.6 3 Maximal “fold-increase” and time (inminutes) to 50% and 90% (t_(0.5) and t_(0.9), respectively) reversal ofthe increases in coronary blood flow caused by various adenosinereceptor agonists. Data represent mean ± SEM of one or two measurementsin each of n pigs.

Example 6

The following example demonstrate the effects of CVT-3146 on thehemodynamic parameters in dogs.

Ten mongrel dogs (weighing 23-27 kg) were premedicated with Acepromazine(0.3 mg/kg im) and anesthetized with sodium pentobarbital (25 mg/kg) andthen intubated and ventilated with room air. A thoracotomy was performedin the left fifth intercostal space using sterile surgical techniques. ATygon catheter (Cardiovascular Instruments, Inc.) was placed in thedescending thoracic aorta for the measurements of blood pressure. Asolid-state pressure gauge (P6.5, Konisberg Instrument, Inc) was placedin the apex of the left ventricle for the measurement of the leftventricular systolic pressure (LVSP) and calculation of first derivativeof left ventricular pressure (LV dP/dt). A Doppler transducer (CraigHartley) was placed on the left circumflex coronary artery formeasurement of CBF. An hydraulic coronary occluder (In Vivo Metric, Inc)was implanted in 4 dogs around the left circumflex coronary artery. Thechest was closed in layers and the catheters and wires were runsubcutaneously and exited in the interscapular area. The dogs wereallowed 10 to 14 days to recover fully from the surgery and were trainedto lie on a laboratory table. The protocols were approved by theInstitutional Animal Care and Use Committee of New York Medical Collegeand conform to the “Guiding Principles for the use and Care ofLaboratory Animals” of the National Institute of Health and the AmericanPhysiology Society.

Arterial pressure was measured by connecting the previously implantedcatheter to a strain-gauge transducer (P23ID, Statham) and mean arterialpressure (MAP) was derived using 2-Hz low-pass filter. LV pressure wasmeasured from the solid-state pressure gauge, and LV dP/dt wascalculated using a microprocessor set as a differentiator and having afrequency response flat to 700 Hz (LM 324, National Semiconductor). Leftcircumflex CBF was measured using a pulsed Doppler flowmeter (System 6,Triton technology), and mean CBF was derived using a 2-Hz low-passfilter. Mean CR was calculated as the quotient of MAP and CBF. Heartrate was monitored from the pressure pulse interval using acardiotachometer (Beckman Instruments). The lead-2 of theelectrocardiogram was recorded during the experiments in order toexamine the alterations in the AV nodal conduction (PR interval). Allsignals were recorded on a direct-writing oscillograph (Gould 2800).

To determine the effects of adenosine and CVT 3146 on CBF and CR inresting dogs baseline hemodynamics and CBF were recorded and then,increasing doses of adenosine: 13, 27, 67, 134 and 267 μg/kg andCVT-3146: 0.1, 0.175, 0.25, 0.5, 1.0, 2.5, 5 μg/kg in 10 ml volumes wereadministered iv for 10 minutes in 10 ml via a catheter inserted into aperipheral vein. Hemodynamics were measured before, during and aftereach dose. Following each dose hemodynamics were allowed to return tobaseline before the administration of the next dose. Changes in heartrate, blood pressure, CBF, and ECG were recorded

The duration of coronary vasodilation was determined using two differentinjection protocols: 1) an iv infusion of 10 ml in 10 seconds and; 2) ivinfusion of 10 ml in 30 seconds. The time to the peak effect in mean CBFincrease and the duration during which CBF remained at least 2 foldabove baseline.

To determine whether tachyphylaxis occurred, three consecutiveinjections of 1 μg/kg CVT-3146 were given as intravenous injections (10ml in 30 seconds) via a catheter inserted into a peripheral vein. Thehemodynamics were allowed to return to baseline between doses.Hemodynamics were measured before, during and after each dose.

Hemodynamic results are expressed as mean±SEM. Data were analyzed usingone way repeated measures analysis of variance, withStudent-Neuman-Keuls post hoc analysis to identify which means weresignificantly (p<0.05) different (Sigma Stat, Version 2.2, JandelScientific, San Rafael, Calif.). To determine the agonist potency fromdose-response curves, doses producing 50% of maximum effect (ED₅₀) werecalculated by fitting curves using the Boltzmann equation. ED₅₀ werecompared using a Student's t-test (p<0.05 being considered assignificant). Because no statistical differences were found among thebaseline values between each dose for each parameter measure, the firstbaseline registered during each experiment was used as the controlvalue. A Student's t-test was used to compare the changes in CBF andhemodynamic parameters produced by 2.5 μg/kg CVT-3146, 267 μg/kgadenosine and 25 μg/kg nitroglycerin (p<0.05 being consideredsignificant).

A dose-response curve of the effect of CVT-3146 on CBF is shown in FIG.11. An IV bolus injection of CVT-3146 caused a dose-dependent increasein mean CBF, with a ED₅₀ of 0.34±0.08 μg/kg and a maximal increase of154±16 ml/min from baseline (45±3 ml/min). In comparison to CVT-3146,adenosine was less potent having an ED₅₀=51±15 μg/kg (p<0.05). Themaximal increase in mean CBF stimulated by CVT-3146 and adenosine weresimilar.

CVT-3146 produced a maximal decrease in CR of 73±2% and 75±2% at 2.5μg/kg at 5 μg/kg, respectively. Adenosine produced a maximal decrease of73±1% at 267 μg/kg (data not shown).

The effects of CVT-3146 and adenosine on left ventricular systolicpressure and dP/dt were compared. Increasing doses of CVT-3146 did notcause significant changes in LVSP (data not shown). In comparison,adenosine increased LVSP at 67 μg/kg, 134 μg/kg and 267 μg/kg, by 12±3%,12±3% and 18±6%, respectively. Both adenosine (267 μg/kg) and CVT-3146(2.5 μg/kg) increased the dP/dt by 29±7% and 39±7% respectively.

Example 7

Example 7 demonstrates the differential effects of CVT-3146 on bloodflow velocity in coronary and peripheral arteries, systemic arterialblood pressure and heart rate in anesthetized dogs.

Mongrel dogs (either sex, 17-21 kg; n=6) were obtained from a localvendor (Barton, Oxford, N.J.). Blood flow velocity in the coronary andcranial circumflex arteries was measured using Doppler transducer-tippedguide wires 0.014″ in diameter (FloWire®, model 1400J) purchased fromCardiometrics, Inc., Mountain View, Calif. For the positioning of theFloWire, a Judkins left coronary guiding catheter (JL3.5, 8F; Cordis)was used. Vascular arterial angiography was performed using Hypaque-76contrast fluid (13racco Diagnostics, Inc., Princeton, N.J.; Lot#9H28899) and a mobile fluoroscopic unit (Philips, BV 29). Systemicarterial blood pressure was measured using an electronictransducer-tipped catheter (Millar). The following pharmacologic. agentswere used: CVT-3146 (CV Therapeutics; Lot #315-53), heparin (SolopakLaboratories, Inc., Elk Grove Village, Ill.; Lot #960211) and sodiumpentobarbital (lot #9700), and acepromazine (lot #3960960), obtainedfrom JA Webster (Fort Dodge, Iowa).

Dogs were sedated with acepromazine (0.25 mg/kg), anesthetized withsodium pentobarbital (30 mg/kg+additional doses (1 mg/kg) given asnecessary to maintain the level of anesthesia), intubated withendotracheal tube and artificially ventilated with room air using arespirator. Following the administration of heparin (20 U/kg+100 U/hr),the pressure transducer-tipped catheter was introduced through the leftfemoral artery and positioned in the descending aorta. The DopplerFloWire was introduced through the right femoral artery. A peripheralvein was cannulated for the administration of all drugs. Doses of 1μg/kg of CVT-3146 and 300 or 200 μg/kg adenosine (Adenosine) were givenmultiple times, once or twice when the Doppler catheter was positionedin a coronary artery and again when the catheter was positioned in thecranial circumflex humeral artery. The sequence of positioning of theDoppler catheter was reversed in consecutive experiments. In each dog,baseline values of measured parameters were recorded following astabilization period of 20 minutes, and CVT-3146 and Adenosine weregiven as intravenous bolus injections (<0.5 ml) followed by aphysiologic saline solution flush (10 ml); the time required for bothinjections of each drug was <15 sec. All parameters were allowed torecover (>30 min) to their respective baseline values between twoconsecutive drug administrations. In all six dogs studied, the effect ofCVT-3146 on coronary artery APV was determined. The effect of CVT-3146on peripheral artery APV was determined in five of the six dogs. Theeffect of adenosine on both coronary and peripheral artery APV wasstudied in five of the six dogs.

Systemic arterial blood pressure (BP) and electrocardiograms (ECG) weremonitored and recorded using Gould Data Acquisition System (model13-4615-65A), a video cassette recorder (Teac, XR 5000) and a chartrecorder (Astromed, 9600). The following parameters were monitored andrecorded: Average peak coronary and peripheral artery blood flowvelocity (APV), mean arterial blood pressure (MAP) (mmHg), and sinuscycle length (SCL; msec). Differences in measure parameters were testedfor statistical significance using ANOVA and Student's t test correctedfor multiple measurements. Data are expressed as the mean±SEM.

CVT-3146 increased APV in the coronary vasculature by 2.6±0.2-fold whileits increase of APV in the peripheral arteries was only 1.1±0.1-fold(Table 7). In contrast the vasodilatory action of adenosine was similarin the two vascular beds: specifically, adenosine increased APV in thecoronary vasculature by 2.5±0.3-fold while its increase of APV in theperiopheral arteries was 2.0±0.4-fold.

TABLE 7 Magnitude of Increases in Coronary and Peripheral Blood FlowVelocity of CVT-3146 and Adenosine in Anesthetized Close-chest DogsAgonist Coronary N Peripheral N CVT-3146 2.6 ± 0.2 6 1.1 ± 0.1 5Adenosine 2.5 ± 0.3 5 2.0 ± 0.4 5 Data are the maximal “folds increase”in average peak velocity (APV) above baseline (APV_(max)/APV_(baseline))after intravenous injection of CVT-3146 (1 μg/kg) and adenosine (200 or300 μg/kg)

The time course of the changes in CBF, PBF, HR and MAP and heart ratecaused by CVT-3146 and adenosine are depicted in FIG. 12 and summarizedin Table 8. The duration of >2-fold increase in coronary APV caused byCVT-3146 and adenosine was <120 sec and <20 sec, respectively; allparameters returned to baseline within 10 min and 5 min post injectionof adenosine and CVT-3146, respectively. Based on the differentialselectivity of the vasodilatory effects of CVT-3146 and adenosine in thecoronary and the peripheral arteries, and the dosage used, CVT-3146 isapproximately 600 times more selective than adenosine in vasodilatingthe coronary vs. the peripheral arterial vasculature.

TABLE 8 Differential effects of CVT-3146 and Ado on blood flow velocity,heart rate and mean arterial blood pressure (MAP) CVT-3146 (1 ug/kg) Ado(200 or 300 ug/kg) Coronary (n = 6) Peripheral (n = 5) Coronary (n = 5)Peripheral (n = 5) Time HR MAP APV HR MAP APV HR MAP APV HR MAP APVBaseline 171 ± 8 128 ± 7 49 ± 7  162 ± 9 116 ± 8 34 ± 8  170 ± 10 126 ±6 42 ± 5  165 ± 10 118 ± 8 33 ± 3 10″ 177 ± 8 126 ± 9 84 ± 4  167 ± 6114 ± 9 34 ± 4 174 ± 7 126 ± 8  90 ± 10 171 ± 6 107 ± 9 62 ± 9 20″ 186 ±9 116 ± 9 106 ± 8  177 ± 7 107 ± 8 38 ± 4  166 ± 11  90 ± 9  85 ± 10 162 ± 15  85 ± 9  76 ± 10 30″ 188 ± 7 117 ± 7 109 ± 10  183 ± 8 111 ± 641 ± 4 173 ± 6 107 ± 9 75 ± 9 168 ± 9  87 ± 9  59 ± 11  1′ 186 ± 7 112 ±7 100 ± 12  182 ± 7 102 ± 6 42 ± 4 175 ± 5 113 ± 6 39 ± 8 171 ± 6 105 ±6 44 ± 7  1.5′ 186 ± 7 110 ± 7 97 ± 11 184 ± 7  99 ± 7 42 ± 4 171 ± 5117 ± 6 55 ± 8 171 ± 6 106 ± 7 38 ± 4  2′ 185 ± 7 110 ± 7 88 ± 11 187 ±8  98 ± 7 40 ± 4 170 ± 5 120 ± 6 52 ± 7 169 ± 6 110 ± 6 35 ± 3  3′ 185 ±7 110 ± 6 81 ± 11 187 ± 8  97 ± 7 40 ± 4 168 ± 8 125 ± 6 49 ± 6 165 ± 9115 ± 7 33 ± 3  4′ 183 ± 8 110 ± 6 75 ± 11 186 ± 8 100 ± 6 39 ± 4  164 ±10 126 ± 6 47 ± 6 160 ± 9 119 ± 8 31 ± 3  5′ 183 ± 8 112 ± 6 72 ± 11 185± 8 101 ± 6 37 ± 5  163 ± 10 128 ± 6 47 ± 6  158 ± 10 121 ± 7 30 ± 2  6′182 ± 8 113 ± 6 65 ± 11  7′ 181 ± 8 115 ± 6 63 ± 12  8′ 180 ± 8 117 ± 655 ± 11  9′ 177 ± 9 119 ± 6 55 ± 11 10′ 176 ± 9 120 ± 6 53 ± 11 Valuesare the mean ± SEM of single determinations in each of the preparations(n).

1. A method of myocardial perfusion imaging of a mammal in need thereof,comprising administering an imaging agent comprising a radionuclide anda dose of a compound that is CVT-3146, also known as(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideto a mammal in need thereof, and determining areas of insufficient bloodflow, wherein the dose is from 0.3 to 103.0 μg/kg, and wherein the dosecauses an increase in peripheral blood flow that is less than 50% of theincrease in coronary blood flow.
 2. The method of claim 1, wherein thedose of CVT-3146 is from 0.3 to 10.0 μg/kg.
 3. The method of claim 1,wherein the step of administering the dose of CVT-3146 takes place by abolus injection of a pharmaceutically acceptable composition thereof. 4.The method of claim 1 further comprising dissolution of CVT-3146 in apharmaceutically acceptable liquid carrier to form a pharmaceuticallyacceptable liquid composition.
 5. The method of claim 1 wherein theCVT-3146 compound is administered in a single dose.
 6. The methodaccording to claim 1 wherein the dose is administered immediately priorto or concurrently with the imaging agent.
 7. The method according toclaim 6 wherein the CVT-3146 compound is administered at the same timeas the imaging agent.
 8. The method according to claim 6 wherein theCVT-3146 compound is administered before the imaging agent isadministered.
 9. A method for increasing coronary blood flow in a mammalin need thereof comprising administering a pharmaceutically effectiveamount of a compound that is CVT-3146, also known as(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide,wherein the pharmaceutically effective amount is from 0.3 to 103.0μg/kg, and wherein the administration causes an increase in peripheralblood flow that is less than 50% of the increase in coronary blood flow.10. The method of claim 9, wherein the pharmaceutically effective amountof CVT-3146 is from 0.3 to 10.0 μg/kg.
 11. The method of claim 9,wherein step of administering the pharmaceutically effective amount ofthe CVT-3146 compound takes place by a bolus injection of apharmaceutically acceptable composition thereof.
 12. The method of claim9 further comprising dissolution of CVT-3146 in a pharmaceuticallyacceptable liquid carrier to form a pharmaceutically acceptable liquidcomposition.
 13. The method of claim 9 wherein the administration of theCVT-3146 compound takes place in a single dose.